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Ligand binding and aggregation of pathogenic SOD1.

Wright GS, Antonyuk SV, Kershaw NM, Strange RW, Samar Hasnain S - Nat Commun (2013)

Bottom Line: Superoxide dismutase-1 mutations decrease protein stability and promote aggregation.We find both compounds interact with superoxide dismutase-1 at a key region identified at the core of the superoxide dismutase-1 fibrillar aggregates, β-barrel loop II-strand 3, rather than the proposed dimer interface site.This illustrates the need for direct structural observations when developing compounds for protein-targeted therapeutics.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biophysics Group, Institute of Integrative Biology, Faculty of Health and Life Sciences, University of Liverpool, Liverpool L69 7ZB, UK.

ABSTRACT
Mutations in the gene encoding Cu/Zn superoxide dismutase-1 cause amyotrophic lateral sclerosis. Superoxide dismutase-1 mutations decrease protein stability and promote aggregation. The mutant monomer is thought to be an intermediate in the pathway from the superoxide dismutase-1 dimer to aggregate. Here we find that the monomeric copper-apo, zinc-holo protein is structurally perturbed and the apo-protein aggregates without reattainment of the monomer-dimer equilibrium. Intervention to stabilize the superoxide dismutase-1 dimer and inhibit aggregation is regarded as a potential therapeutic strategy. We describe protein-ligand interactions for two compounds, Isoproterenol and 5-fluorouridine, highlighted as superoxide dismutase-1 stabilizers. We find both compounds interact with superoxide dismutase-1 at a key region identified at the core of the superoxide dismutase-1 fibrillar aggregates, β-barrel loop II-strand 3, rather than the proposed dimer interface site. This illustrates the need for direct structural observations when developing compounds for protein-targeted therapeutics.

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The structure of monomeric recombinant A4V SOD1 determined by small-angle X-ray scattering.(a) A single size-exclusion chromatogram (see Methods) of monomeric recombinant Cu-apo, Zn-holo A4V SOD1 with radius of gyration (Rg; triangle) and forward scattering intensity (circle) plotted together with absorbance at 280 nm (line). (b) X-ray scattering profile of monomeric A4V SOD1 (solid line with experimental error derived from radial averaging of scattering frames in light gray) compared with the profile of dimeric wild-type SOD1 (dashed line). (c) Distance distribution function of monomeric A4V (solid line) and dimeric wtSOD1 (dashed line) (ref. 60). (d) Space-filling model of monomeric A4V SOD1 and (e) wild-type dimeric SOD1.
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f1: The structure of monomeric recombinant A4V SOD1 determined by small-angle X-ray scattering.(a) A single size-exclusion chromatogram (see Methods) of monomeric recombinant Cu-apo, Zn-holo A4V SOD1 with radius of gyration (Rg; triangle) and forward scattering intensity (circle) plotted together with absorbance at 280 nm (line). (b) X-ray scattering profile of monomeric A4V SOD1 (solid line with experimental error derived from radial averaging of scattering frames in light gray) compared with the profile of dimeric wild-type SOD1 (dashed line). (c) Distance distribution function of monomeric A4V (solid line) and dimeric wtSOD1 (dashed line) (ref. 60). (d) Space-filling model of monomeric A4V SOD1 and (e) wild-type dimeric SOD1.

Mentions: Figure 1a shows the size-exclusion chromatogram of Cu-apo, Zn-holo A4V SOD1 with small-angle scattering parameters plotted together with absorbance at 280 nm. The dimer and monomer species are visible at 3.53 and 3.7 ml, respectively, in both the A280 nm and I(0) plots. Using SEC-SAXS, the radius of gyration (Rg) and maximum intraparticle dimension of dimeric wild-type SOD1 were 20.6±0.3 Å and 61 Å, in comparison with 21.4 Å and 74 Å for dimeric A4V. The A4V monomer is smaller with a radius of gyration (Rg) 17.05±0.3 Å and deduced molecular mass 15.6 kDa28, which equate well with the hydrodynamic radius and molecular mass of monomeric wild-type SOD1 determined by dynamic light scattering (19.4 Å and 17 kDa)29. The X-ray scattering profiles of the Cu-apo, Zn-holo A4V monomer and Cu-apo, Zn-holo dimeric wild-type SOD1 are similar with a characteristic minimum at 2.5 Å−1. The distance distribution functions indicate proteins of approximately equal maximum dimension (Fig. 1b respectively). This result is unexpected given the A4V monomer is half the molecular mass of the wild-type dimer and occurs owing to a single protrusion from the main body of the monomer (Fig. 1d compared with e). This extended conformation is also observed in the dimeric A4V form23, indicating this structural disturbance is independent of oligomeric state.


Ligand binding and aggregation of pathogenic SOD1.

Wright GS, Antonyuk SV, Kershaw NM, Strange RW, Samar Hasnain S - Nat Commun (2013)

The structure of monomeric recombinant A4V SOD1 determined by small-angle X-ray scattering.(a) A single size-exclusion chromatogram (see Methods) of monomeric recombinant Cu-apo, Zn-holo A4V SOD1 with radius of gyration (Rg; triangle) and forward scattering intensity (circle) plotted together with absorbance at 280 nm (line). (b) X-ray scattering profile of monomeric A4V SOD1 (solid line with experimental error derived from radial averaging of scattering frames in light gray) compared with the profile of dimeric wild-type SOD1 (dashed line). (c) Distance distribution function of monomeric A4V (solid line) and dimeric wtSOD1 (dashed line) (ref. 60). (d) Space-filling model of monomeric A4V SOD1 and (e) wild-type dimeric SOD1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3644087&req=5

f1: The structure of monomeric recombinant A4V SOD1 determined by small-angle X-ray scattering.(a) A single size-exclusion chromatogram (see Methods) of monomeric recombinant Cu-apo, Zn-holo A4V SOD1 with radius of gyration (Rg; triangle) and forward scattering intensity (circle) plotted together with absorbance at 280 nm (line). (b) X-ray scattering profile of monomeric A4V SOD1 (solid line with experimental error derived from radial averaging of scattering frames in light gray) compared with the profile of dimeric wild-type SOD1 (dashed line). (c) Distance distribution function of monomeric A4V (solid line) and dimeric wtSOD1 (dashed line) (ref. 60). (d) Space-filling model of monomeric A4V SOD1 and (e) wild-type dimeric SOD1.
Mentions: Figure 1a shows the size-exclusion chromatogram of Cu-apo, Zn-holo A4V SOD1 with small-angle scattering parameters plotted together with absorbance at 280 nm. The dimer and monomer species are visible at 3.53 and 3.7 ml, respectively, in both the A280 nm and I(0) plots. Using SEC-SAXS, the radius of gyration (Rg) and maximum intraparticle dimension of dimeric wild-type SOD1 were 20.6±0.3 Å and 61 Å, in comparison with 21.4 Å and 74 Å for dimeric A4V. The A4V monomer is smaller with a radius of gyration (Rg) 17.05±0.3 Å and deduced molecular mass 15.6 kDa28, which equate well with the hydrodynamic radius and molecular mass of monomeric wild-type SOD1 determined by dynamic light scattering (19.4 Å and 17 kDa)29. The X-ray scattering profiles of the Cu-apo, Zn-holo A4V monomer and Cu-apo, Zn-holo dimeric wild-type SOD1 are similar with a characteristic minimum at 2.5 Å−1. The distance distribution functions indicate proteins of approximately equal maximum dimension (Fig. 1b respectively). This result is unexpected given the A4V monomer is half the molecular mass of the wild-type dimer and occurs owing to a single protrusion from the main body of the monomer (Fig. 1d compared with e). This extended conformation is also observed in the dimeric A4V form23, indicating this structural disturbance is independent of oligomeric state.

Bottom Line: Superoxide dismutase-1 mutations decrease protein stability and promote aggregation.We find both compounds interact with superoxide dismutase-1 at a key region identified at the core of the superoxide dismutase-1 fibrillar aggregates, β-barrel loop II-strand 3, rather than the proposed dimer interface site.This illustrates the need for direct structural observations when developing compounds for protein-targeted therapeutics.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biophysics Group, Institute of Integrative Biology, Faculty of Health and Life Sciences, University of Liverpool, Liverpool L69 7ZB, UK.

ABSTRACT
Mutations in the gene encoding Cu/Zn superoxide dismutase-1 cause amyotrophic lateral sclerosis. Superoxide dismutase-1 mutations decrease protein stability and promote aggregation. The mutant monomer is thought to be an intermediate in the pathway from the superoxide dismutase-1 dimer to aggregate. Here we find that the monomeric copper-apo, zinc-holo protein is structurally perturbed and the apo-protein aggregates without reattainment of the monomer-dimer equilibrium. Intervention to stabilize the superoxide dismutase-1 dimer and inhibit aggregation is regarded as a potential therapeutic strategy. We describe protein-ligand interactions for two compounds, Isoproterenol and 5-fluorouridine, highlighted as superoxide dismutase-1 stabilizers. We find both compounds interact with superoxide dismutase-1 at a key region identified at the core of the superoxide dismutase-1 fibrillar aggregates, β-barrel loop II-strand 3, rather than the proposed dimer interface site. This illustrates the need for direct structural observations when developing compounds for protein-targeted therapeutics.

Show MeSH
Related in: MedlinePlus